TRADITIONAL MEDICI?;,AL PLASTS O F THAILAAND. I. ISOLATION AND STRUCTL-RE ELCCIDSTIOS O F TWO NEW FLAVONOIDS, (2R,3R)-DIHYDROQUERCETIN-4'hlETHYL ETHER AND (2RJ3R)-DIHYDROQVERCETIN4',i-DIhIETHYL ETHER FROM B L UJIEA BALSAMIFERA SIJSIRI RUANGRTSGSI, PIMOLV.\S TAPPATUTHPIJARS and PAYOM T.ISTIV.\T.ISA D e p a r t m e n t of Pharmacognosy, Faculty of Pharniaceufical Sciences, Chiilalongkorn r n i w r s i t y , B a n g k o k , 5 , Thailand and ROBERTP. BORRISand GEOFFREY A. CORDELL D e p a r t m e n t of Pharniacognosg aiid Pharmacology, College of P h a m i a c y , LniFersitg of Illinois at the diedical Center, Chicago, Illinois 60612
AABsTRacT.-The leaves of Blitnzea bnlsczmifera (Compositae) have yielded two new flavonoids, the 4'-methyl ether (la)and the i',i-dimethyl ether (2a) of 2R, 3R-dihydroquercetin. The structures were deduced by spectral interpretation and chemical correlation.
Bliimea balsamifera DC is a member of the tribe Inuleae (Compositae) which has been used in the traditional medicine of some Oriental cultures. The Chinese have used preparations of this plant as a carminative, a mild stimulant, a vermifuge, as a topical application for septic ulcers, and as a preventive medicament in times of epidemics. The ancient Chinese medical literature has recorded its use as a n abortifacient (1).
Preparations of Blumea are presently finding use in the traditional medicine of Thailand, where they are available at local herbal drug shops. Cigarettes are prepared from the chopped, dried leaves of B . balsamifera and smoked t o relieve the pain of sinusitis. An infusion prepared from leaf material is used as a stomachic, carminative, diaphoretic, expectorant and emmenagogue. A decoction of fresh leaves is used, alone or in combination n-ith other plant preparations, as a bath for n-omen follon-ing childbirth. The chemistry of Blumea constituents has been of some interest for a t least 65 years. The essential oil was the first product to be studied (2). I n this initial study, d-carvotanacetone, 1-tetrahydrocarvone, a mixture of butyric, isobutyric and u-octanoic acids, and an unidentified phenol were isolated. Subsequently, I-borneol (l), fenchone (3), 1&cineol ( 3 ) , two carvotanacetone derivatives ( A ) , a diester of coniferyl alcohol (5), some polyacetylenes and thiophene derivatives (5, 6), campesterol ( i ) stigmasterol , ( 8 ) , sitosterol (9), xanthoxylin (l), erianthin and 5,3',4'-trihydroxy-3,6,7-trimethoxyflavone (3, 10-12), other unidentified flavonoids (1, l o ) , coumarins and triterpenes (I), and myristic acid (3) have been isolated from Blumea species. The present study was undertaken in order to expand our knowledge of the constituents of this medicinally interesting genus, and describes the isolation of two flavonoid derivatives, (2R,3R)-dihydroquercetin-4'-methylether (la) and (2R,3R)-dihydroquercetin-4',i-dirnethyl ether (2a), both of which are new natural products. 541
542
Journal of S a t u r a l Products
[Vol. 44, s o . 5
EXPERIMENTAL1 PLANT M.ATERI.~L.--L~~~material of Blumea balsamifera D C (Compositae) was purchased from a local herbal drug shop in Bangkok and authenticated by comparison with herbarium specimens a t the Botany Section, Technical Division, Department of Agriculture, hlinistry of Agriculture and Cooperatives, Thailand. This material was then air dried and coarsely powdered.
EXTRACTIOX A N D INITIAL FR.4cnoNaTIo.l-.-The air dried and powdered plant material (4.5 kg) was macerated twice for 5 day periods with 95% ethanol (20 and 15 liters). The ethanol extracts were pooled, the alcohol removed in F U C U O , and the residue suspended in 3 liters of 10% ethanol. After filtration, the filtrate was treated with a 10% aqueous lead acetate solution until no further precipitation occurred. Further filtration then afforded a clear solution which was extracted with chloroform (3 x 7 liters). The combined chloroform extract was dried (NazS04)and then evaporated in ~ l a c u ot o yield 13.5 g of a brown syrupy mass which, when stirred with chloroform (90 ml), produced a pale yellow precipitate. Recovery b y filtration and drying yielded a solid (Fraction A, 977 mg) and a filtrate which was evaporated in tsacuo to a yellow syrup (Fraction B, 12 9). S E P A R A ~OFO FRACTIOS X .&.--Thin layer chromatography (tlc) of Fraction -4 (silica gel GZ, chloroform: acetone (9:l) ) indicated the presence of only two components (Rf=0.17 and 0.35). The total fraction was divided into four portions. Each portion was dissolved in chloroform (2 ml), adsorbed onto silica gel ( 5 g), dried, then placed on top of a dry silica gel 602 column (2.5 x 40 cm), and eluted with chloroform-acetone (9:l). Fractions of 20 ml each were collected and compared by tlc. Those fractions of similar composition were combined. This procedure produced 3 major fractions which were evaporated in 'r'ucuo. Fractions 1-5 yielded no residue on evaporation; fractions 6-10 were homogeneous by tlc and were designated PS-2; fractions 11-19 were also homogeneous by tlc and were designated PS-1. (2R,3R)-DIHYDROQUERCETIX-4'-I\IETHTL ETHER (la).-Fraction Ps-1, when evaporated t o dryness in '~'ucuo,yielded 766 mg (1.7 x 10--4yc)of a light yellow, amorphous powder, mp 173-4"; [a]24D+14.9";ir, vmax (KBr), 3400, 1645, 1600, 1280, and 1160 em-': uv, data summarized in table 1; cd (RleOH), [8]32e=+3.35xlo', [8]zs3.5=-1.31x lo5,[O]253.5=+1.95xlo;, [8]220=+1.44x 106 deg.cm2jdmole; 1H-nmr (DMSO-de) 6, 3.78 (3H, s, ArOCH3),4.48 ( l H , bd, J = 1 1 Hz, 3-H), 3.81 (3H, m 3 x OH), 5.03 ( l H , d, J = l l Hz, 2-H), 5.87 (2H, AB system, 6-H and 8-H), 6.91 (3H, bm, 2'LH, 5'-H and 6'-H), and 11.88 ( l H , bs, OH); 'H-nmr (acetone-de) 6, 3.88 (3H, s, ArOCHa), 4.61 (lH, d , J = l l . 5 Hz, 3-H), 5.08 (IH, d, J = l l . 5 Hz, 2-H), 5.98 (2H, AB system, 6-H and 8-H), 7.00-7.10 (3H, m, 2'-H, 5'-H and 6'-H), and 11.68 ( l H , bs, OH); ms, m / z 318 (hl-, 3753, CI6H,,O7),289 (43), 166 (50),165 (26), 164 (36), 163 (loo), and 137 (39). (2R,3R)-DIHrDRO?rERCETIN-4',7-DIMETHTL ETHER (2a).-Fraction PS-2, when evaporated t o dryness in cucuo, yielded 94 mg (2.1 x 10-5Cc)of a light yellow, amorphous powder, mp 164-7"; [ ~ ~ ] ~ ~ ~ + 1ir,4 .vmax 8 " ; (KBr), 3480, 1630, 1510, 1273, 1160, and 1130 cm-l; uv, data summarized in table 1. cd (MeOH), [8]311.5=+3.59 x lo5, [O]2-,=-1.26 x lofi, [ 8 ] 2 5 3 5=+1.71 x lo', [ O ] ~ Z5 =~ +1.28 x 106 deg.cmz/dmole; 1H-nmr (DMSO-ds) 6,3.79 (GH, bs, 2 x ArOCHa), 4.55 ( I H , m, 3-H), 5.09 ( l H , d, J = l l Hz, 2-H), 6.10 (2H, ilB system, J = 2 Hz, 6-H and 8-H), and 6.95 (3H, m, 2'-H, 5'-H and 6'-H): 1H-nmr (acetone-de) 6, 2.78 (3H, bm, 3 x OH), 3.86 (3H, s, ArOCH3), 3.87 (3H, s, ArOCH3), 4.62 (lH, d, J=11.6 Hz, 3-H), 5.11 ( l H , d, J=11.6 Hz, 2-H), 6.07 (2H, AB system, G-H and 8-H), and 7.00-7.10 (3H, m, 2'-H, 5'-H and 6'-H); ms, m l z 332 (If+, 25%, CliHI6Oi),303 (36), 179 (16), 167 (loo), 166 (29), 164 (35), 151 (20), and 137 (23).
OXIDATIOX OF la.--A sample (15 mg) of la was suspended in 2N H z S O ~ ( 5 ml) and heated on a steam bath under a gentle stream of air for 24 hours. After the sample was cooled to room temperature and extracted with ethvl acetate (4 x 5 ml), partition was effected against a saturated aqueous solution of sodium Gcarbonate to remove residual acid. The organic phase, when dried ( S a z S 0 4 )and evaporated in cucuo, yielded 14.2 mg (95%) of a dark yellow, waxy solid, 3a; uv, Xmax (RleOH), 369, 292, 270 sh, 255 and 205 nm; Xmax (MeOHfSaOCHB), 388, 326, 277 and 205 nm, R-ith no change for at least 24 hours. Welting points were determined on a Kofler hot plate and are uncorrected. The uv spectra were obtained with a Beckman model DB-G spectrophotometer. The ir spectra were determined with a Perkin Elmer model 283 spectrophotometer; adsorption bands are reported in wave numbers (cm-1). 1H-nmr spectra were recorded with a T-arian T 4 0 A instrument operating a t GO MHz, with a Nicolet hlodel TT-7 Fourier Transform attachment. Tetramethylsilane was used as an internal standard, and chemical shifts are reported in 6 (PPlM). Mass spectra were obtained with a Yarian MAT-112s double-focusing spectrometer operating a t 80 eV and 220". Optical rotations were obtained in methanol with a Perkin Elmer model 241 polarimeter. CD spectra were obtained in methanol with a JASCO model J-40A automatic recording spectropolarimeter. ZE. hlerck, Darmstadt, W. Germany.
Sep-Oct 19811 Ruangrungsi et al. : Flavonoids of Blumea balsamifera
543
TABLE 1. Ultraviolet spectra d a t a of PS-1 and PS-% PS-1 (la)
PS-2 (2a)
MeOH
327 sh 290 235sh 206
(log e=3.89) (4.48) (4.54) (4.82)
327 sh 287 230sh 216sh 205
(3.76) (4.49) (4.57) (4.68) (j.81)
SaOCH3
327 290sh 250sh 206
(4.62) (4.03) (4.09) (4.82)
327sh 290 230sh 2lish 207
(3.79 (4.44) (4.57) (4.70) (4,79)
TaOAc . . . . . . . . . .
327 294sh
327sh 287
(3.76) (4.49)
NaOAc+H3B03.,
327sh 290
327sh 287
(3.76) (4.49)
BlC13.. . . . . . . . . . .
381 315 282 223 206
382 315 287sh 225 206
(3.92) (4.57) (4.11) (4.70) (4.92)
AlCla+HCl
377 315 282 223 206
384 312 287sh 225 206
(3.89)
(4.50) (4.22) (4.68) (4.91)
aA411UT- spectra were recorded using the standard procedures given in reference 13.
TRI~~ETHTLSILTLITIOS OF 3a.-A sample (14 mg) of 3a was reacted with TRI-SIL3 (3 ml) for 15 minutes. The solvent and excess reagent, when removed under vacuum (oil pump) a t room temperature, yielded 22 mg (82%) of a crude product which displayed 1H-nmr (CC1,) 6, 3.87 (3H, S, A4rOCH3),6.13 ( l H , d , / = 2 H z , 6-H), 6.29 ( l H , d, J = 2 Hz, 8-H), 6.84 ( l H , d, J=8.5 Hz, 5'-H), and 7.57-7.75 (2H, m, 2'-H and 6'-H). OXIDATIOSOF 2a.-A sample (15 mg) of 2a was suspended in 2ru' H2S0, ( 5 ml) and heated on a steam bath under a gentle stream of air for 48 hours. After the sample was cooled t o room temperature and extracted with ethyl acetate (4 x 5 ml), partition was effected against a saturated aqueous solution of sodium bicarbonate t o remove residual acid. The organic phase, when dried (Sa2S04) and evaporated i n I ' U C U O , yielded 12.4 mg (83%) of a vellow waxv solid, 4a; uv, hmax (hleOH), 364, 288, 260, 213 and 208 nm; Xmax (hleOH+KaOCH3), 413, 333, 290, 230 and 211 nm, with no change for at least 24 hours. STRCCTVRE ELUCIDATIOS OF la A X D 2a.-The proton nmr data of isolate PS-1 strongly suggested that it was a dihydroflavonol derivative, and the characteristic 11 Hz doublets a t 4.48 and 5.03 ppm indicated a trans-diaxial arrangement of 2-H and 3-H in such a system. T h a t the signal for 3-H was further split and, on addition of D20, sharpened to a doublet indicated that the hydroxyl at C-3 was not methylated (13a). The AB system centered at 5.87 ppm, showing mela coupling of approximately 1 Hz, suggested that they were protons a t C-6 and C-8 in a dihydroflavonol oxygenated at C-5 and C-7 (13b). The pronounced bathochromic shift of the band I1 absorption in the uv spectrum of PS-1 on addition of sodium acetate indicated the presence of a free hydroxyl at C-7 (13c). Addition of aluminum chloride reagent produced bathochromic shifts of band I (54 nm) and band I1 (25 nm) characteristic of a second free hydroxyl at C-5 (13d). hlass spectral peaks at nzlz 166 and 137 substantiated that the remaining hydroxyl and the methoxyl group were indeed on the B-ring (14, 15). 3Pierce Chemicals, Rockford, Illinois, USA.
Journal of Natural Products
544
=
la
R
lb
R = CH,,
H,
[Vol. 44, No. 5
R': C H ~
2a
R=H,
R'= H
2b
R = C H 3 , R'= H
R'= CH,
OR
OR
P
O
R
'
O OH
0
3a
R=H,
R'- CH,
4a
R = H,
R'-CH,
3b
R-CH,,
R'= H
4b
R = CH3,
R'= H
The multiplet a t 6.91 (DMSO) or 7.00-7.10 (acetone) in the nmr spectrum of PS-1 are suggestive of oxygenation of C-3' and C-4' (13e). However, definitive assignment of the substitution pattern could not be made on the basis of these spectra. A portion of the isolate was oxidized (2N H,SOa/air) (16) to the corresponding flavonol, which should be either tamarixetin (3a) or isorhamnetin (3b). Comparison of the nmr spectrum of the TMS ether of this unknown flavonol with published spectra of the two reference compounds (13f)showed that the unknown flavonol corresponded to tamarixetin (3a). This was further substantiated b y the stability of the uv spectrum of the oxidation product in the presence of sodium methoxide. Flavonols having a free hydroxyl a t C-4' are unstable in sodium methoxide solution, and their uv spectra degenerate in a few minutes (13g). The flavonol produced by the oxidation of PS-1 was seen to be stable in the presence of sodium methoxide for a t least 24 hours. Also, tlc comparison of the oxidation product with isorhamnetin (3b)showed them to be different in several systems. A sample of tamarixetin was, unfortunately, not available. The absolute stereochemistry a t C-2 and C-3 was determined by means of circular dichroism. Comparison of the cd curves of PS-1 with those of dihydroflavonols of known configuration (17) established the absolute configuration as 2R,3R, the signs of the Cotton effects from 400 to 200 nm being respectively -, +, Thus PS-1 (la) is assigned the structure (2R,3R,)-dihydroquercetin4'-methylether (or (2R,3R)-dihydrotamarixetin). The nmr spectrum of the isolate PS-2 was identical to that of PS-1 except that it showed the loss of one exchangeable proton and the addition of a second aromatic methoxyl group. Since the uv spectrum was unchanged on addition of sodium acetate, this methoxyl group is located a t C-7 (13c). Mass spectral evidence confirmed that the additional 14 mass units were indeed in the A-ring. Sulfuric acid oxidation of PS-2 was used to establish the B-ring substitution pattern as in 2a rather than 2b. Thus, the uv spectrum of the oxidation product 4a was found to be quite stable in sodium methoxide solution, and tlc comparison with a reference sample of rhamnazin (4b) showed4a to be different in several systems. Circular dichroism again established the absolute configuration to be 2R,3R. Thus, PS-2 (2a)is assigned the structure (ZR,3R)-dihydroquercetin4',7-dimethylether. I n 1972, Herz et al. (18) reported the isolation of a dihydroflavonol from a Eupatoraum hybrid (Compositae) having either 2a or 2b as its structure. They were unable t o completely define their isolate, having lost the remaining sample in an unsuccessful oxidation reaction.
+,
+.
Sep-Oct 19811 Ruangrungsi et al. : Flavonoids of Blumea balsamifera
545
Comparison of the aromatic region of the nmr spectrum (in DMSO) of their isolate with that of PS-2 showed them clearly t o be different. Therefore, from the data presented in ref. 18 and comparison with our isolate, PS-2, we can now propose the structure of the Eupatorzum isolate as (2R,3R)-dihydroquercetin-3’,7-dimethyl ether (2b), which is also a previously unreported natural product. ACKNOWLEDGMESTS The authors are indebted t o Professor H . Wagner, Institut fur Pharmazeutische Biologie Universitat Jlunchen, West Germany, for authentic samples of rhamnazin and isorhamnetin; t o Professor W . Herz, The Florida State University, Tallahassee, Florida, USA, for copies of the ’H-nmr spectra of the dlhydroflavonol from E u p ~ t ~ r i uspecies; m t o Mr. D. hlcPherson. Department of Pharmacognosy and Pharmacology, College of Pharmacy, UIMC, Chicago. for obtaining the mass spectral d a t a , and to Mr. Jerry Lasky, Department of Medicinal Chemistry, College of Pharmacy, UIJIC, Chicago, for help in obtaining the optical rotations and cd spectra. One of the authors (R.P.B.) wishes to express his gratitude t o the -4merican Foundation for Pharmaceutical Education for its support in the form of a graduate fellowship and t o the Graduate College, UIJIC, Chicago, for its support in the form of the Dorothea H . Fleming Student Research Fellowship. Receired 12 January 1981
LITERATURE C I T E D 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
T.C . Kong, S. \-. Hu, F. K . Lau, C. T. Che, H . W. Yeung, S. Cheung and J. C. C. Hwang, A m . J . Chinese Med., 4(2), 105 (1976). J . L. Simonsen and AI. G . Rau, J . Chem. Soc., 121, 876 (1922). W.Karrer, “Konstitution and 1-orkommen der Organischen Pflanzenstoffe,” Birkhauser Verlag, Basel, 1976, p. 130, 217, 222, 292 and 625. F . Bohlmann, C. Zdero and A. G. Ramachandran S a i r , Phytochemistry, 18, 1062 (1979). F Bohlmann and C. Zdero, Tetrahedron Letts., 69 (1969). F. Bohlmann, T . Burkhardt and C . Zdero, “Saturally Occurring Acetylenes,” Academic Press, S e w York, S,17., 1973, pp. 350-1. R. Pal, 6 . K . Moitra, S . S . Chakravarti and R . S . ildhya, Phytochemistry, 11, 1855 (1972). H . K. Desai, D . H . Gawad, T. R. Govindachari, B. S. Joshi, I-. Ti. Kamat, P . C. Parthasarathy, K . S. Ramachandran, RI. S . Shanbhag, -4. R. Sidhaye and 9. Viswanathan, Zndznn J . Chem., 13, 97 (1975). H . K . Desai, D . H . Gam-ad, T. R. Govindachari, B. S. Joshi, P. C. Parthasarathy, K . S. Ramachandran. K. R. Ravindranath, A. R . Sidhare and N. 1-iswanathan, Zndzan J . Chem., 14b, 473 (1976). C. B. Rao, T . S . Rao and B. Muralikrishna, Planta X e d . , 31, 235 (1977). P . K . Bose and P. Dutt, J . zndzan Chem. Soc., 17, 45 (1940). P . K . Bose, A . K . Barua and P . Chakrabarti, J . Zndzen Chem. Soc., 45,851 (1968). T. J . Mabry, K. R. hlarkham and XI. B. Thomas, “The Systematic Identification of Flavonoids,” Springer 1-erlag, Xew Tork, S.T.,1970, (a) p. 267; (b) p. 262; (c) p. 170; (d) p. 171, (e) p. 326-7; ( f ) p. 298-9; (9) p. 47. J. B. Harborne, T . J . JIabry and H . hlabry, “The Flavonoids, P a r t 1,” Academic Press, 1975, p. 105. Xew Tork, S.l-., E . Rodriguez, X . J . Carman, G. Van der 1-elde, J. H . RlcReynolds, T. J . Mabry, M. A. Irwin and T. A. Geissman, Phytochemistry, 11, 3509 (1972). G. Pavanasasivam and 11.U. S.Sultanbawa, J . Chem. 5’06. Perkzn Trans. I , 612 (19i5). W.Gaffield, Tetrahedron, 26, 4093 (1970). W.Herz, S.Gibaja, S.\-. Bhat and A. Srinivasan, Phytochemistry, 11, 2859 (1972).
